ALK status is clinically pivotal for determining eligibility
ALK status is clinically pivotal for determining eligibility for ALK-directed targeted therapy. As current gold standard for ALK rearrangements detection, Vysis ALK Break Apart fluorescence in situ hybridization (FISH) kit (Abbott Molecular, Abbott Park, IL) was approved by U.S. Food and Drug Administration (FDA) in 2011 . The Ventana ALK (D5F3) IHC assay, another routine diagnostic method using a D5F3 rabbit monoclonal primary antibody to detect aberrant ALK protein expression, has also been authorized by U.S. FDA as a companion diagnosis of Ceritinib . Nevertheless, the best laboratory method to determine ALK status in lung cancer remains controversial for their own limits. The advent of next-generation sequencing methods (NGS) has facilitated high-throughput molecular analysis for a mass of genes in a single test, providing a cost- and tissue-efficient alternative to simultaneously detect gene alterations including copy number alterations, deletions, insertions, rearrangements, and single-base substitutions [, , ]. To date, ALK assessment by NGS methods has not been clinically validated, and the concordance with traditional methods is not well established.
Discussion Hitherto, consensus has not been achieved regarding the best detection approaches for ALK fusion gene. Detection of ALK rearrangement by FISH is currently the gold standard, which has been validated in a series of clinical trials [9,23]. Nevertheless, in our study, 11 of 45 (24.4%) cases evaluated by FISH had inconclusive results for technical reasons like weak or no green signal detected or isolated R signal, implying FISH assays could be technically challenging and complicated to interpreting, probably due to the subtlety of the emitted signal and the topographic location of these genes in the chromosome . The possibility of signal decay after long-term tissue block storage might account for it . Secondly, patients with advanced NSCLC usually only provide small biopsy. Sometimes it is difficult to ensure that more than 50 lung cancer CY7-SE are interpreted. Thirdly, intratumoral heterogeneity, which was reported to coexist with histologic heterogeneity in both single-driver and ALK/EGFR coaltered LADCs, may result in the discrepance. Altered oncogenic drivers in spatially separated subclones of the same tumor may be different . Among nine FISH-inconclusive cases using Crizotinib, 9/9 were IHC ALK + and 7/9 were NGS ALK+, and the overwhelming majority (8/9) achieved response to Crizotinib, indicating an omission of potential targeted therapy population by FISH. While in 21 FISH-defined cases, 3 of 4 FISH negative cases were proved PR to the targeted therapy and 1 was evaluated as SD. It was in part similar to the study of Ali et al., which identified 11 FISH ALK negative cases in 31 NGS ALK positive patients (35%) and found 7 of 11(64%) FISH ALK negative cases sensitive to Crizotinib . Other studies that have applied NGS to confirm inconsistent IHC and FISH results also implied that FISH could deny patients’ access to ALK inhibitors for a significant false-negative rate [, , , ]. Hence, the combination of ALK fusion detection method would achieve higher accuracy. The borderline cut off values, as Camidge et al. confirmed that when >4 fields were counted, the >15% cell positivity cut point would ensure maximal sensitivity and specificity, affected the detection results from another perspective . The high costs, time consuming, specific equipment, and trained personnel also make it not feasible for routine use. The correlation between ALK IHC staining and FISH detection has been extensively reported in the literature [33,34]. Among several available ALK IHC antibodies, Clone D5F3 (Cell Signaling Technology) has been evaluated by several groups. Minca et al. concluded that IHC using the D5F3 IHC antibody was more informative than FISH, including adding more ALK-positive cases . Marina et al. claimed that IHC (D5F3 antibody) provided excellent sensitivity and specificity (100% and 97.7%, respectively), suggesting a promising alternative to FISH . In our study, Ventana-D5F3 IHC assay was adopted and it presented the highest positive rate (94.5%) among three methodologies. Characteristics like time-saving, accessible and economical also ensure the foundational role in ALK screening .